Identifying horizontal transfers, recombination events and test
of endosymbiont similarity:
To test for horizontal transmission of endosymbionts across the soil
arthropod hosts, two sets of analyses were done. The first was a visual
estimation of the horizontal transfer obtained by comparing host and
endosymbiont phylogenies. The second was a quantitative estimation of
the correlation between the pairwise distance matrices of hosts and
their corresponding endosymbionts. The Spearman method (r ) of
Mantel test for correlation (Legendre & Legendre, 2012) was determined
in R v1.2.5. A total of 5 different correlations were computed with
their corresponding host sequences: all the Wolbachia infected
samples (33), only A supergroup Wolbachia infected samples (16),
only B supergroup Wolbachia infected samples (15), onlyCardinium infected samples (8) and Arsenophonus infected
samples (7).
To identify and visualize recombination events within theWolbachia concatenated MLST sequences (2079bp), SplitsTree
v4.14.8 (Huson, Kloepper, & Bryant, 2008) was used to construct
phylogenetic network using uncorrected p -distance and
Neighbor-net method (Bryant & Moulton, 2004). To statistically evaluate
presence of recombination, Φ test (Bruen, Philippe, & Bryant,
2006) was used in SplitsTree v4.14.8. These identified events were then
evaluated using RDP4 v4.97 (Martin, Murrell, Golden, Khoosal, & Muhire,
2015) which has several suits of programs to detect and identify
recombination events (like RDP, GENECONV, MaxChi, SiScan, BootScan,
Chimaera and 3Seq). A recombination event was considered significant if
it was shown to be statistically significant by at least three or more
detection methods. Recombination breakpoints were also identified with
RDP v4.97.
To test whether the endosymbionts are largely being transferred within
the community, and thereby have less sequence divergence than expected,
we used the model by Baldo et al. (2008) to test the similarity of
endosymbionts within community. Briefly, the mean pairwise distance of
endosymbionts presents in soil arthropod community were compared with an
equivalent number of pairwise distances randomly selected from the
database for 10000 iterations. These iterations were computed to give a
null distribution for comparison with the soil endosymbiont sequence
data by Wilcox rank-sum test with continuity correction and 95%
confidence interval (performed in R v1.2.5). From our sampling, we found
17 unique ST’s for Wolbachia supergroup A and 15 forWolbachia supergroup B infections. Whereas, in the PubMLST
database there were 228 unique ST’s for A supergroup and 252 ST’s for B
supergroup (last checked May 2019). For the 9 samples infected withCardinium and 8 for Arsenophonus , 248 and 228 homologues,
respectively, were obtained from NCBI (last checked May, 2020). Pairwise
distance was calculated using MEGA7 (Kumar et al ., 2015) and then
corrected with Jukes and Cantor model in
DNAsp v5.10.01 (Librado & Rozas,
2009). Density plots for
endosymbiont divergence from the soil arthropods and the databases were
plotted in R (http://R-project.org).